Since flowers having new traits are always favored in the flower industry, the development of such flowering plants is industrially important. Breed improvement based mainly on crossing has produced species with a variety of traits. However, in the crossing-based breed improvement, available gene resources are restricted to crossable closely-related plants, and thus colors and traits that can be introduced are limited in most cases, and besides it is rare that single plant species have various colors and traits. In terms of color for example, it was impossible to produce blue roses and carnations, yellow morning glories or geraniums by the crossing-based breed improvement.
However, the use of the gene recombinant technology can realize the development of a variety of traits by introducing various genes into any plants transcending the species barrier and by artificially altering plant metabolisms etc. For example, there is an example wherein in roses and carnations that cannot produce delphinidin in the flower petal, the gene of the flavonoid 3′,5′-hydroxylase enzyme that is required to synthesize delphinidin was expressed to produce delphinidin, which enabled the creation of blue flowers that are not present in nature (Tanaka 2006).
However, in Japan, the research and development, cultivation, distribution etc. of such artificially-created gene recombinant plants are required to abide by the regulations set forth in “Law Concerning the Conservation and Sustainable Use of Biological Diversity through Regulations on the Use of Living Modified Organisms” (Cartagena Protocol). In other countries as well, field cultivation etc. of gene recombinant plants have been regulated based on similar laws. Specifically, for plants whose pollens are fertile and for which a horde of crossable closely-related plants occur in Japan, evaluation on crossability, or on the possibility of gene proliferation from a recombinant plant to a closely-related wild species, is obligatory.
In the case of roses for which a horde of closely-related species occur in Japan and which are multiflorous and fructiferous, quantities to be analyzed on the presence of crossing between cultivated and wild species become enormous, and thus the establishment of simple and accurate analytical technologies is being sought after. So far, the one that utilizes microsatellites as the molecular marker has been reported (Debener 2003, 2006). Furthermore, analysis may also be carried out with an identification method based on ploidy determination using flow cytometry. However, any of the above methods had problems that they lacked accuracy, versatility and/or simplicity. Furthermore, since cultivated roses today were created by artificially crossing about 8 wild species, it was not easy to obtain DNA markers that can distinguish cultivated species from wild species.
In accordance with the present invention, the KSN gene, a target gene, is a gene involved in the perpetual blooming of roses obtained from Rosa chinensis spontanea, and the gene was created by inserting an about 9 kb transposon into the KSN gene of a one season flowering rose. It is reported that the insertion of a transposon serves to inhibit the expression of said gene, which led to the deregulation of anthogenesis control at the shoot apex promoting anthogenesis, which resulted in the perpetual blooming nature (Iwata et al., Japanese Unexamined Patent Publication (Kokai) No. 2006-149202).
It is already elucidated that roses of the cultivated species contain, in the homologous configuration, said gene having a transposon inserted therein. On the other hand, roses of the wild species have, in the homologous configuration, a KSN gene which, in principle, does not contain a transposon.
As used herein, Rosa chinensis is one of the wild species that became an ancestor of cultivated roses, and a one season flowering rose. R. chinensis spontanea is a mutant lineage thereof and a perpetual blooming rose.
Patent document 1: Japanese Unexamined Patent Publication (Kokai) No. 2006-149202.